Ixodes scapularis, is the arthropod vector for Borrelia burgdorferi, the causative agent of Lyme disease. In addition, the tick serves as a vector for other bacterial, protozoan and viral pathogens. There is an unmet need for safe and effective vaccines against these pathogens. Co-infected ticks can transmit more than one pathogen simultaneously. Vaccines targeting the tick would have the advantage of blocking multiple tick-transmitted diseases. While Anaplasma phagocytophila is transmission commences after about 24 h of tick feeding, B. burgdorferi transmission begins after 24-36 h of tick feeding. To develop vaccines that can block transmission of multiple pathogens, it is crucial to thwart feeding and transmission at the outset of tick feeding. Acquired tick-immunity is a phenomenon wherein repeated infestation of vertebrate hosts results in tick rejection within 12-24 h of tick attachment. Tick-immunity also effectively abrogates transmission of B. burgdorferi. Host immunity directed against tick salivary antigens crucial for establishing tick-feeding contributes to this phenomenon. Acquired tick-immunity that is apparent within the first 24 h of attachment must therefore be directed against early antigens. Research to date has focused largely on tick salivary antigens expressed 3-4 days after tick attachment, with little success. The focus of this proposal is, therefore, to identify tick salivary antigens expressed and secreted in the first 24 h of tick attachment that are targeted by host tick-immunity. It is expected that by defining antigens that evoke tick-immunity, we will identify salivary antigens that can be targeted to effectively block tick feeding at the very outset and consequently block the transmission of pathogens harbored by the tick. The emphasis in this proposal will be on B. burgdorferi transmission. An immunoscreening approach will be taken to identify 24 h tick salivary antigens that react with 24 h nymphimmune serum. The physiological role of these antigens in tick feeding and in B. burgdorferi transmission will be determined using the RNA interference strategy. The antigens will be prioritized based on their role in feeding and Borrelia transmission and vaccine candidates determined. Results obtained will provide the basis for future vaccine development studies. [unreadable] [unreadable] [unreadable]